The following relates to the biological assay arts. It is described with particular reference to example embodiments that relate to detection of rare cells, such as epithelial cells, in the buffy coat of a centrifuged blood sample. However, the following relates more generally to identifying fluorescent dye-tagged cells in a biological fluid layer using a fluorescent imager, in which there is less than about one dye-tagged cell per optical field-of-view of the fluorescent imager.
In the technique of quantitative buffy coat analysis, a whole blood sample is drawn and processed using anti-coagulant additives, centrifuging, and so forth to separate the blood into components including a buffy coat component comprised principally of white blood cells. Rare cells of interest which are present in the buffy coat, such as certain epithelial cells associated with certain cancers, are tagged using a suitable fluorescent dye, and fluorescent imaging is then used to count the fluorescent dye-tagged cells of interest. Such quantitative buffy coat analysis is a promising non-invasive technique for screening for certain pathologies. For example, some cancer screening techniques have been proposed which are based on quantitative buffy coat analysis.
The concentration of rare fluorescent dye-tagged cells in the buffy coat is low—a typical fluorescent imager optical field-of-view may not contain any fluorescent dye-tagged cells at all. This makes it difficult to focus the fluorescent imager. Additionally, typical substrates or sample holders for optical viewing, such as glass slides or test tubes, have surface height variations substantially exceeding the depth-of-view of typical fluorescent images. Thus, as the field-of-view of the fluorescent imager is scanned the buffy layer sample may go into and out of focus, and the optical imager should be frequently refocused. Moreover, depending upon how the buffy coat layer sample is mounted, it may be thicker than the depth-of-view of the fluorescent imager. Fluorescent dyed-cells that are outside of the depth of view will not appear as sharp, well-defined features in the fluorescent images. In view of these difficulties, locating fluorescent dye-tagged cells in the fluorescent images is challenging.